The ultimate goal of the research proposed here is to elucidate the mechanism of ATP hydrolysis and ATP synthesis by the ATP synthase, an enzyme common to all energy transducing membranes. Alterations in the ability or efficiency of these coupled reactions may be an important factor in aging and can explain some of the cases of mitochondrial myopathy, encephalopathy and cardiopathy that are being reported with increasing frequency in the medical literature. Work in this laboratory began with the bovine heart ATP synthase but has switched to the enzyme from Escherichia coli. The bacterial enzyme has many of the structural features and functional properties of the heart enzyme but can be manipulated genetically and can also be dissociated into component subunits and then reassociated into a functional complex. The major approach being used is electron microscopy of specimens embedded in a thin layer of ice (cryoelectron microscopy). A three-dimensional structure of the ECF1 has been obtained at low resolution (25A). A collection of monoclonal antibodies (mAbs) have been purified against the various subunits of the complex and these are being used to decorate ECF1 and ECF1F0. The antibody-antigen complex can then be examined by electron microscopy and the locus of individual subunits in the structure identified. The structural changes that accompany functioning of the enzyme complex are also being studied. ECF1 shows cooperative ATPase activity, i.e. binding of substrate ATP, in one catalytic site increases the off-rate of product ATP in another site. In ECF1F0, this cooperativity is important in both ATP hydrolysis and ATP synthesis, and these reactions are coupled to proton pumping through the F0 part. Protease digestion, mAb binding, chemical crosslinking and cryoelectron microscopy are all being used to examine the nature of the conformational changes that occur during cooperative ATPase activity and in coupling of this reaction to proton pumping.